Int. J. Mol. Sci. 2015, 16, 17193-17230; doi:10.3390/ijms160817193 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Protein Folding and Mechanisms of Proteostasis José Fernando Díaz-Villanueva, Raúl Díaz-Molina and Victor García-González * Facultad de Medicina, Universidad Autónoma de Baja California, Mexicali, Baja California 21000, México; E-Mails: [email protected] (J.F.D.-V.); [email protected] (R.D.-M.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +52-68-6557-1622 (ext.121); Fax: +52-68-6557-1622 (ext.109). Academic Editor: Salvador Ventura Received: 1 April 2015 / Accepted: 11 June 2015 / Published: 28 July 2015 Abstract: Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis. Keywords: proteins; folding; proteostasis; misfolding OPEN ACCESS
38
Embed
Protein Folding and Mechanisms of Proteostasis...unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Int. J. Mol. Sci. 2015, 16, 17193-17230; doi:10.3390/ijms160817193
International Journal of
Molecular Sciences ISSN 1422-0067
www.mdpi.com/journal/ijms
Review
Protein Folding and Mechanisms of Proteostasis
José Fernando Díaz-Villanueva, Raúl Díaz-Molina and Victor García-González *
Facultad de Medicina, Universidad Autónoma de Baja California, Mexicali, Baja California 21000,
(PKR-like ER kinase), and ATF6 (activating transcription factor 6). Synthesis of ER resident
chaperones and folding catalysts, is induced to increase the folding capacity; and global mRNA
translation is attenuated to decrease the folding load [123]. Likewise, through a process called
ER-associated degradation (ERAD) misfolded proteins could be retained in the ER and retrotranslocated
into the cytosol for proteasomal degradation [124]. UPR operates within the context of a translocation
machinery that is compartmentalized between cytoplasm and ER [125].
Int. J. Mol. Sci. 2015, 16 17203
ATF6 is a transcription factor that is initially synthesized as an ER-resident transmembrane protein
bearing a large ER-luminal domain. Upon accumulation of unfolded proteins, ATF6 is packaged into
transport vesicles that pinch off the ER and deliver it to the Golgi apparatus [126]. ATF6 is processed
by two proteases, S1P and S2P (site-1 and site-2 protease), that sequentially remove the luminal
domain and the transmembrane anchor, respectively [127,128]. The liberated N-terminal cytosolic
fragment, ATF6 (N), then moves into the nucleus to activate UPR target genes involved in protein
folding, such as BiP, the protein disulfide isomerase, and GRP94 (glucose-regulated protein 94), a
chaperone of the Hsp90 family [14].
The second branch of the UPR is coordinated by PERK. When it is activated upon sensing ER
stress, PERK oligomerizes and phosphorylates itself and the ubiquitous translation initiation factor
eIF2α, then indirectly inactivates eIF2 and inhibits the global translation of mRNA, conditions that
ameliorate the ER stress [14]. Concomitantly, translation of the transcription factor ATF4 is induced,
which promotes transcription of two important target genes, CHOP (transcription factor C/EBP
homologous protein) and GADD34 (growth arrest and DNA damage-inducible 34). CHOP is a
transcription factor that controls genes encoding components involved in apoptosis. GADD34 encodes
a PERK-inducible regulatory subunit of the protein phosphatase PP1C that counteracts the action of
PERK by dephosphorylating eIF2α [14,129,130]. Then, this pathway has an intrinsic feedback regulation.
The best-studied branch of UPR is IRE1, which transmits UPR signaling through a bifunctional
transmembrane kinase/endoribonuclease that splices mRNA through a non-conventional mechanism.
Binding of unfolded proteins triggers conformational changes following lateral oligomerization on the
ER membrane, which in turn activates the IRE1 ribonuclease activity. Furthermore, IRE1 processes
the mRNA of transcription factor XBP1 (X-box binding protein 1) [14] (Figure 3). This active,
processed form of the transcription factor XBP1s controls expression of genes with X-box elements in
their promoters, genes encoding ER chaperones and folding catalysts [131–134]. Additionally, the
IRE1/XBP1 pathway is essential to activate genes which carry out ERAD functions [135], promoting
the development of an elaborate ER response [136]. Active conformation of the kinase domain of
IRE1 has been revealed through its crystal structure, to be an oligomeric assembly [122]. Indeed
ribonuclease activity of IRE1 has been described to be proportional to the extent of IRE1
oligomerization [137].
UPR represents a focal point where different sources of stress converge, and stress signaling is
coordinated within tissue hierarchies and further integrated [14]. Several activated transcription factors
generated by UPR enter the nucleus and activate the production of their target genes, this mechanism
establishes a feedback loop that relieves the ER stress by supplying more ER protein-folding capacity
according to cell requirements [138]. In addition to linear information flow, the three branches of UPR
transmit information to each other through a phenomenon known as cross-talk to fully integrate the
signaling networks [139]. Nodes of interaction and communication between proteins required for
cellular function should be highly regulated.
More experimental evidence is needed to fully understand specific thresholds necessary for the
activation of stress signaling pathways, and in turn when homeostasis is reached again, the mechanisms
that allow these responses to be turned off. For instance, IRE1 and PERK signaling duration is critical to
determine the fate of cells during prolonged stress [140]. Therefore, when homeostasis cannot be
Int. J. Mol. Sci. 2015, 16 17204
re-established, UPR switches from a protective mechanism to a cytotoxic response [141], indeed UPR
can function as an apoptotic executor which decreases cell viability (Figure 3).
Taking into account that ER must manage folding and modification of proteins in concentrations
surpassing 100 mg/mL [142], recognition of unfolded proteins must be a highly precise mechanism
to initiate the correct cellular response. Additionally, other mechanisms constitute a stress adaptation
pathway that could reestablish homeostasis of proteins.
Figure 3. Activation of the inositol requiring enzyme 1 (IRE1) branch of the unfolded
protein response (UPR) pathway is tightly controlled. (A) Schematic representation of
IRE1 oligomerization and cellular response induced by unfolded proteins. The structure
employed was obtained from the protein data bank (PDB) access code: 3fbv;
(B) Uncontrolled protein aggregation in the formation of amyloid fibrils; (C) Effect of the
overactivation of IRE1 branch on cellular homeostasis. Adapted from reference [141];
(D) Structural representation of IRE1 with a focus on the surface of interaction between
monomers. Inset: sterol binding site, with two bound molecules of quercetin. Structure was
obtained from PDB access code: 3LJ0.
3.3. Spatial Compartmentalization of Protein Folding
Connections between loss of proteostasis, protein aggregation phenomenon, and conditions ranging
from ageing to neurodegeneration, underscore the importance of the knowledge of the mechanisms
that cells employ to manage protein misfolding [142]. For instance, it is estimated that up to 15% of
Int. J. Mol. Sci. 2015, 16 17205
nascent chains in human cells are co-translationally tagged for degradation, which emphasizes the
importance of co-translational degradation in protein quality control at the ribosome [143,144]. Cells
must not only promote accurate folding but also must prevent the accumulation of misfolded species
that may arise from inefficient folding, errors in translation, and aberrant mRNAs [145].
An important condition to maintain the functionality of cells is associated with localization of
misfolded or aggregated proteins into specialized compartments that are distinct from the organelles.
Cytotoxicity is avoided through the confinement of misfolded proteins, aggregates, or amyloid like
structures within appropriate and specific subcellular compartments, thus avoiding the subsequently
nucleation of protein aggregates. In this sense, cells have developed mechanisms to solubilize and
fold these proteins, when possible, leaving degradation in defined quality control compartments as a
last-resort mechanism [146].
When quality control machineries fail, such as those previously mentioned, protein-controlled
sequestration into specific compartments represents an alternative cellular defense against proteotoxic
stress [147–149]. Upon proteasome impairment, misfolded proteins are distributed into spatially and
functionally distinct compartments. Evidence has revealed a conserved sequestration of ubiquitinated
proteins into membrane-enclosed juxtanuclear compartments, such as JUNQ (juxtanuclear quality
control compartment). JUNQ is a cellular quality control space wherein soluble misfolded proteins
accumulate for refolding or proteasomal degradation [150].
Under proteotoxic conditions in a cell, several chaperones and proteasome complexes can be
located surrounding the JUNQ compartment, suggesting that JUNQ allows the concentration of
misfolded proteins with chaperones, therefore increasing the probability of refolding, as opposed to
simple uncontrolled degradation [151]. Substrates targeted to JUNQ are primarily soluble proteins,
which are rapidly exchanged with the surrounding environment. When native folding is not reached,
JUNQ substrates are ubiquitinated and recruit proteasome components, triggering protein degradation.
In an important manner, when these mechanisms fail or are diminished, protein misfolding has been
observed by the formation of amyloid fibrils [152,153]. In parallel, diverse strategies have evolved to
maintain the native structure of proteins for prolonged periods of time, and avoiding their conversion
into non-functional misfolded structures [154].
Distinct cytoplasmic structures, spatially distant from JUNQ have been observed to contain large
and highly insoluble aggregates [150]. These compartments denominated IPODs (insoluble protein
deposits) contain insoluble aggregates and amyloid like-fibrils; whereas multiple IPODs can exist at
the same time in cytoplasm, only a single JUNQ is found within each cell. These types of compartments,
underscore their essential function in sequestering proteins and triaging them to re-establish native
protein folding or initiate protein degradation.
While misfolded polypeptides of JUNQ are sequestered in a detergent soluble state, and the
aggregated polypeptides are retained in IPOD [142], other compartments have an important function;
Q-bodies whose formation and processing depend on the cortical ER. The maturation and clearance of
Q-bodies require chaperones Hsp70 and Hsp90 [142].
Biochemical functions of chaperones and their spatial localization within the cell are fundamental to
understand folding impairments during pathological states [142,155], and considering the presence of a
dynamic relationship between damaged and aggregated proteins, the function of these compartments
Int. J. Mol. Sci. 2015, 16 17206
could be very important to maintain the proteostasis in the first instance, and collectively cell
homeostasis [156].
3.4. Proteasome, Structure, and Function
Nascent or newly synthesized polypeptides are predisposed to a high quality control process
associated to their folding, avoiding the accumulation of anomalous proteins. Most studied systems to
maintain proteostasis, are performed by molecular chaperones, as well as two mechanisms of protein
degradation, the ubiquitin-proteasome system and the lysosomal proteolysis through autophagy [157–159].
Proteasome (26S) is a multimeric complex whose function is protein degradation through its
endoprotease activity. Proteasome acts primarily on short-lived proteins with regulatory functions and
on misfolded proteins. Protein degradation is a specific and efficient process, which depends on
ATP. It is involved in functions such as modulation of cell cycle, apoptosis, and cell differentiation,
response to extreme temperature changes, oxidative stress, immune responses, genetic regulation, and
metabolism [160,161].
The 26S proteasome is directly associated to protein degradation via the ubiquitin system. It is
composed of a catalytic subunit comprising a cylindrical central structure with proteolytic activity,
denominated 20S proteasome. Additionally, two regulatory subunits 19S are present, which have
ATPase activity; these are involved in recognition and elimination of ubiquitin chains. Likewise, 19S
subunits participate in the unfolding of client proteins [160] (Figure 4).
The 20S catalytic subunit consists of four complexes arranged in the manner of a ring, forming a
hollow cylindrical structure. Each ring consists of seven different protein subunits, two rings consist of
subunits denominated α (α1–7) located at the ends of the cylindrical structure, the other two rings
consist of subunits called β (β1–7) and are located in the central area [162]. The N-terminal domains of
α-subunits occlude access to the interior of the proteasome, while three of the β subunits have a
protease activity. Therefore, the 20S subunit is a structure α1–7, β1–7, β1–7, α1–7, with proteolytic capacity
and highly conserved between eukaryotes [163,164].
The 19S regulatory subunit is a complex consisting of at least 19 protein subunits, which are
distributed to form the base and lid of the 19S subunit. The base is formed by six proteins with ATPase
activity (Rpt1–Rpt6) that are in contact with α-subunits of the 20S proteasome, and four proteins
lacking ATPase activity (Rpn1, 2, 10, 13). The protein subcomplex which makes up the lid is
integrated by proteins without ATPase activity (Rpn3–9, 11, 12), but contains binding sites for
recognition of ubiquitinated proteins, and maintain a function of deubiquitinase [162,165,166].
Int. J. Mol. Sci. 2015, 16 17207
Figure 4. Scheme of the 26S proteasome. Proteins that make up the base and lid of the 19S
regulatory subunit are shown. The cylindrical portion of the 20S catalytic subunit is shown
in an open conformation, showing the arrangement of α and β proteins identified in orange
and blue, respectively (Adapted from [167]).
The client proteins must have at least four ubiquitins attached to be recognized. Recognition and
anchoring occurs through Rpn10 and Rpn13, which associate with polyubiquitin. Rpn11 accomplishes
the remotion of ubiquitin chains. Proteins with ATPase activity in the 19S subunit perform the
unfolding of ubiquitinated proteins while interacting with the α proteins of the 20S subunit, a condition
that allow its opening, and leads to client proteins into the 20S subunit [163,168,169].
Proteolytic activity of the 20S proteasome subunit lies in the extreme N-terminus of the subunits β1,
β2, and β5 of β-rings. β1 subunit has a caspase activity on amino acids, β2 shows the trypsin activity
on basic amino acids, and β3 subunit a chymotrypsin activity on hydrophobic amino acids. Even more,
in vitro experiments have shown that the 20S subunit alone can present proteolytic activity, generating
peptides of 3–15 residues [167].
Alzheimer’s, Parkinson’s, and Huntington’s diseases are characterized by dysfunction of the
ubiquitin-proteasome system, and accumulation of misfolded proteins in the central nervous system.
Particularly, in Alzheimer’s disease, the patient has two important lesions, extracellular amyloid
plaques, and intraneuronal neurofibrillary tangles formed by Aβ peptide [170,171], which is generated
through sequential cleavage of amyloid precursor protein (APP). In vitro experiments showed that
Aβ40 directly binds to the inside of 20S proteasome and selectively inhibits its chymotrypsin-like
activity [172,173]. More recent evidence shows that Aβ42 also impairs proteasome activity [174,175];
both of them may be endogenous inhibitors of the proteasome. This condition is a consequence of
dysfunction of the ubiquitin-proteasome and its possible association to disease.
Int. J. Mol. Sci. 2015, 16 17208
3.5. Autophagy Mechanism
Autophagy includes a lysosomal degradation pathway, in which cells self-digest their own
components, and has been shown to be essential for survival, differentiation, development, and
homeostasis. Autophagy involves the sequestration of cytoplasmic components in double membrane
autophagosomes, wherein these structures fuse with lysosomes and their cargoes, and are delivered for
degradation and recycling [176].
Mechanisms of autophagy play an important role in removing protein aggregates and organelles that
fail to be degraded by the ubiquitin-proteasome system [177]. Furthermore, the role of autophagy in
maintaining macromolecular synthesis and ATP production is likely a critical mechanism underlying
its evolutionarily conserved pro-survival function [178]. For instance, when cells suddenly undergo a
surge in metabolic demand, autophagy may be needed to generate sufficient intracellular metabolic
substrates to maintain energy levels. This self-digestion process not only provides nutrients to maintain
cellular functions during fasting, but can also relieve cells of superfluous or damaged organelles,
misfolded proteins, and invading microorganisms [179]. Indeed, it has been described that autophagy
could provide an adaptive role to protect organisms against diverse pathologic conditions; which
include: cancer, neurodegeneration, aging, and heart disease [180]. Through this process, cells carry out double-membrane vesicles, denominated autophagosomes, which
could sequester organelles, proteins, or portions of the cytoplasm for delivery to the lysosomes [159].
The core pathway of mammalian autophagy begins with the formation of an isolation membrane
(also called a phagophore), and involves a minimum of five molecular components, including (1) the
AuTophaGy related 1 (Atg1)/unc-51-like kinase (ULK) complex; (2) the Beclin 1/class III
phosphatidylinositol 3-kinase (PI3K) complex; (3) two transmembrane proteins, Atg9, and vacuole
membrane protein 1 (VMP1); (4) two ubiquitin-like protein conjugation systems (Atg12 and
Atg8/LC3); and (5) proteins that drive fusion between autophagosomes and lysosomes. It has been
described that some of these core autophagy pathway components are directly modulated by cellular
stress signals [181,182].
Several functions of autophagy, such as, elimination of defective proteins and organelles, prevention
of protein aggregate accumulation, and clearance of large poly-ubiquitinated proteins, overlap with
those of the ubiquitin-proteasome system; however, pathways leading up to autophagy are uniquely
capable of degrading entire organelles such as mitochondria, peroxisomes, ER, as well as intact
intracellular microorganisms [183]. Unlike proteasomal degradation, the autophagic breakdown of
substrates is not limited by steric conditions, because substrates do not need to be unfolded to pass
through the narrow pore of the proteasomal barrel. Oligomeric and aggregated proteins are poor
substrates for proteasomal degradation, and better targets for autophagic degradation; therefore,
preventing the intracellular accumulation of misfolded proteins and contributing to the proteostasis [183].
Autophagy is also upregulated when cells undergo remodeling events, such as developmental
transitions or to rid themselves of damaging cytoplasmic components, during oxidative stress or
infections [182]. Likewise, autophagy is activated as an adaptive catabolic process in response to
different forms of metabolic stress, including growth factor depletion and hypoxia [183]. Through
autophagy, bulk degradation generates free amino acids and fatty acids that can be recycled or further
processed to maintain ATP production in cells when it is required [182].
Int. J. Mol. Sci. 2015, 16 17209
Nevertheless, alterations in autophagy could result in the accumulation of ubiquitinated and
aggregated proteins, and in turn damaged organelles. In experimental diseases, the self-cannibalistic or,
paradoxically, even the prosurvival functions of autophagy may be deleterious [182]. Autophagosomes
have been observed to accumulate in the brains of patients with diverse neurodegenerative diseases,
including Alzheimer, transmissible spongiform encephalopathies, Parkinson, and Huntington [182].
For instance, autophagosomes accumulate in dystrophic neurons of Alzheimer’s disease patients,
possibly as a result of impairment in autophagolysosomal maturation, consequently contributing to the
accumulation of pathogenic Aβ peptide [184].
On the other hand, the pharmacological activation of autophagy reduces the levels of soluble and
aggregated conformations of mutant huntington, mutated proteins in spinocerebellar ataxia, as well as
mutant forms of α-synuclein and tau. This activation reduces cellular toxicity and their neurotoxicity in
mouse and Drosophila models [185]. Particularly in these models, neuroprotection modulated by
autophagy may be due to a quantitative reduction in the amounts of toxic protein species as well as
anti-apoptotic effects [185].
Autophagy can influence life and death decisions of cells, being cytoprotective or self-destructive;
and being directly linked to apoptotic death pathways. Based on the knowledge of physiological
functions of autophagy, it has been determined that both, basal levels of autophagy and stress-induced
increase of autophagy, are likely determinant in mammalian homeostasis [186].
4. Clinical Focus
Several regulatory and control strategies have evolved in biological systems to protect the
phenomenon of protein folding. Molecular chaperones, protease activities, and molecular factors work
together to refold or remove proteins [187]. When these defensive housekeeping systems of cells are
unable to counteract with these challenges and homeostatic systems are gradually deteriorated,
pathological conditions associated with misfolding become evident [11,90,188]. Even without genetic
defects, protein translation is sufficiently error-prone to allow a missense mutation in proteins
every 1000 to 10,000 amino acids, resulting in defects between 4% and 36% of all new proteins
synthesized [189–191]. This can be tolerated if these proteins can be degraded, but when the load is
excessive, as occurs during cell stress, cell death could appear [192,193].
4.1. Neurodegenerative Diseases
Mechanisms preventing amyloid fibril formation are associated with properties of cell environment,
including the location of proteins within specific compartments [150,194], as well as the presence of
molecular chaperones and degradation mechanisms, such as the ubiquitin–proteasome system and
autophagy [195–199] (Figure 2). Protein misfolding is developed when proteins are unable to attain or
maintain their biologically active conformation [187].
Protein misfolding and formation of toxic aggregates, for instance could affect the proteostasis in
cells to induce ER stress, and in this condition, UPR is required. Likewise, molecular chaperones can
target specific steps in the process that leads to misfolding, specifically inhibiting either primary or
secondary nucleation processes [200], such as the case of the Hsp70 function [201].
Int. J. Mol. Sci. 2015, 16 17210
The disturbance of proteostasis can lead to a situation that is considered a metastasis in proteins,
therefore initial aggregation events trigger a cascade of pathological processes that could mark the
progression of chronic-degenerative diseases [202]. In cases such as Aβ peptide, α-synuclein, and
others peptides, the direct connection between misfolding and the formation of amyloid fibrils, is
determined by structural transitions at the molecular level [40]. Therefore, amyloid formation is
triggered when the protective mechanisms have been exceeded, or due to malfunction of mechanisms
of cell regulation [40].
Protein aggregation and amyloid formation are two fields that have been extensively studied by the
association of amyloid deposition with a range of chronic degenerative disorders, from Alzheimer’s
disease (AD) to diabetes mellitus type 2, many of which are major threats to human health and welfare
in the modern world [203,204]. AD is characterized by cognitive alterations, memory loss, and
behavioral changes. Amyloid plaques and neurofibrillary tangles are the hallmark lesions in the
pathology and both arise from protein misfolding phenomena [205]. In this condition, the Aβ peptide
and tau protein suffer conformational changes that span disordered states and lead to maturation of
toxic aggregates. The presence of such structures leads to ER stress, activating IRE1 and PERK
pathways that active CHOP, conditions reported to induce neuronal death [120]. Likewise, amyloid
precursor protein (APP) and presenilins 1 and 2 have been associated with a familiar form of
Alzheimer. Taking into account their ER membrane location, stress conditions may alter the activity of
presenilin 1 and 2, inducing an increase in the processing of Aβ peptide [206]. In Parkinson disease,
the death of dopaminergic neurons and protein aggregation (Lewy bodies) in different regions of the
brain, is present [207].
4.2. Metabolic Diseases
ER stress is associated with inflammatory and stress signaling pathways, which could exacerbate
metabolic dysfunction, contributing to obesity, insulin resistance, fatty liver, and dyslipidemia [208,209].
The presence of amyloid structures in pancreatic islets of Langerhans is a pathophysiological condition
related with diabetes mellitus type 2. These deposits are composed of a peptide hormone named
amylin [210]. Amylin is normally soluble, and its structure in the monomeric state is natively
disordered. However, secondary structure transitions can be important to attain the three-dimensional
structure found in amyloid fibrils. Aggregation of amylin is associated with an increased response of
ER stress, which leads to dysfunction of pancreatic β cells, apoptosis, and eventually the loss of the
cell mass of islets [211–214]. Likewise, high levels of plasmatic non-esterified fatty acids can
contribute to β cell dysfunction [215]. In our work group, we have been able to establish a potential
relationship of interaction between these important biomolecules [216].
UPR is chronically activated in atherosclerotic related cells, particularly on advanced lesional
macrophages and endothelial cells [217]. Oxidative stress, oxysterols, high levels of intracellular
cholesterol, and saturated fatty acids, are conditions that can lead to prolonged activation of the UPR in
advanced lesions. Likewise, these arterial wall stressors may be associated with obesity, insulin
resistance, and diabetes, all of which promote the clinical progression of atherosclerosis. The potentially
important proatherogenic effect of prolonged ER stress is activation of inflammatory pathways. Even
Int. J. Mol. Sci. 2015, 16 17211
more, prolonged ER stress triggers apoptosis in macrophages, which in turn leads to plaque necrosis if
the apoptotic cells are not rapidly cleared [217].
Conditions associated to atherosclerosis, such as chronic ER stress, affects systemic risk factors at
the level of hepatic lipid metabolism and pancreatic β-cell function [218]. A specific focus has been
performed on signaling modulation through IRE1. Recent developments in understanding how IRE1α
functions to promote cell death versus cell survival at a protein structural level, raise the possibility of
several specific drugs that can block IRE1α-dependent cell death [219,220].
4.3. Cancer and Protein p53
Factors which contribute to a significant increase in protein misfolding incidences are mutations,
thermodynamics, and external stress conditions [187]. The proposal that ER stress signaling could
either be beneficial for tumor growth or play a guardian role to prevent cell transformation is very
important to analyze [221]. Tumor cells are often subjected to major molecular changes due either to
transformation-dependent metabolic demand or to stressful environments, including hypoxia, nutritional
stress or pH stress [222]. For example, one of the conditions of activation of UPR in cancer, has been
attributed to the hypoxic condition in the tumor surrounding environment [223].
Involvement of PERK and IRE1 arms of the UPR in tumor growth has been broadly
characterized [222]. In these conditions, ER stress signaling represents an important constituent of
tumor progression and survival [222]. IRE1α enhances angiogenesis and may alter cell adhesion and
migration through regulated IRE1-dependent decay (RIDD). Likewise, cells deficient in XBP1 or
PERK have a large reduction in their ability to form solid tumors in mice models. In fact, negative
regulation of chaperone activity has been investigated as an anticancer strategy [224,225].
Expression of components of the ER protein-folding machinery, such as BiP, has also been suggested
to promote tumor progression, cell survival, metastasis, and resistance to chemotherapy [226]. Strategies
to downregulate BiP in models of cancer or through the use of inhibitors of the ATP-binding domain
have great cytotoxic potential [227–232].
On the other hand, p53 is a transcription factor with an essential role in guarding cells responses to
various stress signals, through the induction of cell cycle arrest, and apoptosis as well as effects that
are independent of its ability to transactivate gene expression [233]. Mutation of the tumor suppressor
p53 is the most frequent genetic alteration in human cancer [234]. The majority of the mutations occur
in the DNA-binding domain of the p53 (residues 102–292), which result in loss of DNA binding.
Zinc binding, coordinated by H179, C176, C238, and C242, is critical for maintain the native folding of
p53 and requires reduction of thiol groups on cysteines. Residues from the loop-sheet-helix motif
interact in the major groove of the DNA, while an arginine from one of the two large loops interacts in
the minor groove. Loops and the loop-sheet-helix motif represent the conserved regions of the core
domain, and contain the majority of the p53 mutations identified in tumors [235]. In this sense, several
mutations induce conformational changes in the DNA binding surface [236] although destabilized
mutants of p53 can be stabilized by the binding of other molecules [237,238].
Consider that cellular and extracellular spaces are highly saturated environments that allow a wide
variety of interactions between molecules. This feature, often referred to as macromolecular saturated
environment could have important consequences in the thermodynamics of molecules, affecting the
Int. J. Mol. Sci. 2015, 16 17212
conformational states of proteins [239], and then proteostasis. Even more, according to scientists
working in different fields of knowledge, nature appears to have employed disorder to create high
levels of organization. In some cases nature seems to have created disorder, when there is, in the first
place a lack of it [240]. This situation extrapolated to the role of proteins and their association with
disease, could find their origin in the way proteins carry out many structural changes, employing finely
tuned disorder-to-order transitions [34].
5. Protein Folding in Drug Development
In all organisms, energy and nutrient management requires the highly regulated and coordinated
operation of many homeostatic systems. Much of the development and evolution of these systems
has taken place in a different environment to the one we now experience as modern humans,
which includes excess nutrients, new dietary components, lack of physical activity, and an increased
life span [209]. In fact, the requirements for the timespan as well as the magnitude of adaptive
responses have dramatically increased due to rises in life expectancy and a chronic lifetime exposure to
the stress signals [209].
Opportunities for the development of effective therapies against protein-aggregation disorders lie in
the discovery of molecules that decrease the concentrations and formation rates of anomalous protein
assemblies or that enable our natural defenses to maintain their efficacy for longer periods of time [40].
A key milestone in the development of any new therapy is the selection of appropriate molecular targets.
5.1. Strategies Focus on Amyloidosis
Pharmacological strategies for effective therapy in the treatment of diseases associated with protein
folding, might consider the following conditions: inducing the stabilization of native state, reducing the
concentration of aggregation-prone species, blocking the nucleation and growth of aggregates; in
general conditions that are able to reduce the risk of aggregation. Another strategy is optimization of
cell defense mechanisms to maintain their efficacy for longer periods of time, using molecules that can
act as pharmacological chaperones. As already suggested, the most effective procedures for the
prevention and treatment of misfolding diseases, are likely to be those that address the earliest events
in their development [92,241].
Stability of proteins and design of sequences that can efficiently acquire a globular structure,
have been considered to be one of the factors that prevent the conversion of a globular protein into
amyloid-like fibrils. It has been demonstrated that loss of stability in the native state is a primary
mechanism by which mutations promote their pathogenic effects in some hereditary forms of
amyloidosis [242]. For example, nature has used strategies to reduce the number of patterns that favor
the formation of β-structure, alternating groups of hydrophobic and hydrophilic residues, as well as
maintain a high net charge in the sequence, place strategically charged amino acids through the
sequence, generate short β-strands on the edges of large β-sheets, incorporate proline residues, and
cover β-sheets with α-helices [81,83,243,244].
A representative example in this topic is the drug tafamidis (Fx-1006A), a small molecule that acts
as a pharmacological chaperone of transthyretin (TTR), stabilizing the native TTR and some variants.
TTR tetramer dissociation has been described as the rate-limiting step in the amyloid formation
Int. J. Mol. Sci. 2015, 16 17213
cascade. Tetramer dissociation is followed by dimer dissociation yielding unstable monomers. TTR
monomers easily unfold leading to spontaneous self-assembly into amyloid fibrils [245]. Tafamidis is the
first disease modifying pharmacological treatment available to treat familial amyloid polyneuropathy.
In fact, fibril formation has been demonstrated as a mechanism for sequestration of oligomeric species,
in a way that cells reduce toxicity [246].
5.2. Chemical Chaperones
Identification of molecule regulators of the UPR signaling as potential therapeutic strategies to treat
protein misfolding and other human diseases, results in a promising approach. UPR is considered
a target for drug discovery because of emerging evidence from animal models indicating its contribution
to diverse diseases, including cancer, metabolic diseases, diabetes, neurodegenerative disorders,
inflammation, liver dysfunction, and brain and heart ischaemia [247].
Development of drugs that interfere with ER stress, have wide therapeutic potential. Five groups of
strategies according to their mechanism of action have been characterized: compounds directly binding
to ER stress molecules, chemical chaperones, inhibitors of protein degradation, antioxidants, and drugs
affecting calcium signaling. Treatments are generally inhibitory, also lead to increased viability, except
when applied to cancer cells [248].
Chemical chaperones are described as low-molecular mass compounds that stabilize the folding of
proteins and buffer abnormal protein aggregation. In this case, chemical chaperones have been shown
to improve ER function, through diminishing protein misfolding events. The most studied chemical
chaperones in a disease context are 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA),
which have been approved by regulatory authorities for primary biliary cirrhosis (4-BPA) and urea
cycle disorders (TUDCA) [249].
In animal models of obesity, chemical chaperones reduced ER stress in the liver of mouse,
improved insulin sensitivity and glucose homeostasis [250], and reversed leptin resistance [251].
Treatment with 4-PBA also improved glucose tolerance in patients with insulin-resistance [252], and
TUDCA partially restored insulin sensitivity in liver and muscle [253].
High-throughput screening for IRE1 modulators has identified plant-derived flavonols as activators
of IRE1 sensors, as well as possible new regulatory sites of interaction. Docking of small-molecule
libraries suggests the presence of a pocket associated with dimerization/oligomerization of IRE1
including a binding site to sterols, which could represent an important binding site for the regulation of
IRE1 signaling [254,255]. Comparative and systematic studies are needed in a better way to define
the real therapeutic value of manipulating ER stress levels, in addition to outlining possible side
effects [209].
Likewise, approaches through compounds that bind and stabilize mutants of p53 have been
performed. Upon screening of a library of over 100,000 compounds and further optimizing of the hits,
compound 7 (CP-31398) was shown to promote the conformational stability of wild-type p53
DNA-binding domain and that of full-length p53 [237,256]. Screening of the diversity set from the
National Cancer Institute led to the discovery of some chemical chaperones with mutant
p53-reactivating capacity: compound 854 known as PRIMA-1 (p53 reactivation and induction of
massive apoptosis) and compound 954 known as MIRA-3 (mutant p53 dependent induction of
Int. J. Mol. Sci. 2015, 16 17214
rapid apoptosis). Additional screening of small compound libraries also identified compound 1055
denominated STIMA-1 (SH group-targeting compound that induces massive apoptosis) [187].
5.3. Final Considerations
Three-dimensional structure of proteins in general, and specifically proteins that participate in UPR
could provide critical information for the development of new pharmacological treatments, this
approach may be incomplete, since the disordered domains of proteins involved in UPR and
chaperones, have been proven to be critical for their function. Likewise, complex pathways to ensure
proteostasis in different subcellular compartments, defined as unfolded protein responses have evolved
in the cytosol and mitochondria, which are finely coordinated and require close communication with
the nucleus [248]. Even more, the UPR has demonstrated impact on various immune cells, in which
it regulates the secretion of pro-inflammatory cytokines and innate immunity signals [254]. These
conditions reflect the complexity of cell physiology that might be considered for drug development.
Whereas cellular and extracellular spaces are highly saturated environments [97,98] which allow a
wide variety of interactions between molecules, not all biologically active compounds have the desired
physicochemical properties to be a drug, which must be sufficiently lipophilic to be absorbed, maintain
polar properties to cross the gastrointestinal wall, and have a vulnerable chemical functionality,
then molecules can be targeted by liver catabolic systems [26]. Without doubt a more complex
understanding of the threshold of responses that occur within cells to sustain the proteostasis, implies a
greater understanding of the regulatory mechanisms that regulate protein folding, which will result in
an increase in tools that could be the basis to modulate the functional activity of therapeutically
important proteins.
6. Conclusions
Mechanisms that modulate protein folding within the highly saturated cellular environment, which
are regulated by a highly sophisticated network of communication between proteins, reflect the
complexity of cellular processes. These features span several molecular hierarchies, from the use of
small disordered regions within intricate three-dimensional structures, and an effective folding
phenomenon to keep hidden the hydrophobic domains. Likewise required are the activity of highly
organized molecules such as chaperones, and the participation of pathways associated with complete
degradation of organelles, to maintain the homeostasis of proteins as a whole. Evidence indicates that
nearly all large, subcellular processes, from the organelles to ribosomes, may have specific ways of
sensing the proteome and reacting to proteotoxic stress, and so that every step in the life of proteins is
under close scrutiny. Therefore, the insights into the features of the functional conformations of
proteins, the environments in which they work, and the ways that cellular defense mechanisms
normally function so effectively together to maintain protein homeostasis, can expand the possibilities
for better treatments against human diseases.
Int. J. Mol. Sci. 2015, 16 17215
Acknowledgments
The authors recognized the help of Julia Dolores Estrada Guzmán, and we want to thank
Nadia Gutiérrez Quintanar and Pedro Alberto Méndez Jaime for editorial support. This work was supported
by Coordinación de Posgrado e Investigación, Universidad Autónoma de Baja California, and by Programa
para el Desarrollo Profesional Docente para el tipo Superior, Secretaria de Educación Pública.
Author Contributions
Victor García-González conceived the idea and wrote the manuscript; José Fernando Díaz-Villanueva
was involved in discussion and helped of manuscript preparation; and Raúl Díaz-Molina critically
reviewed the manuscript. All authors approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Badyaev, A.V. Stress-induced variation in evolution: From behavioural plasticity to genetic
assimilation. Proc. Biol. Sci. 2005, 272, 877–886.
2. Nussinov, R. The spatial structure of cell signaling systems. Phys. Biol. 2013, 10, 045004.
3. Nussinov, R.; Wolynes, P.G. A second molecular biology revolution? The energy landscapes of